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Screening method for enzyme spectrum of kinase of tumor inhibitor

A tumor suppressor, kinase zymography technology, applied in the field of determination or inspection of C12Q containing enzymes or microorganisms, can solve the problems of slow speed, human body damage, low sensitivity, etc., and achieve the effect of fast speed, large detection scale and high sensitivity

Inactive Publication Date: 2014-05-21
EAST CHINA NORMAL UNIV
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Problems solved by technology

Among them, the radioisotope-based method has the following disadvantages: a large amount of radioactive waste is produced; the half-life of radioactive ATP is very short, only 14.3 days, so new isotopes need to be ordered frequently; the radioisotope method is not suitable for high-throughput or ultra- High-throughput screening; very strong cross-interference phenomenon; relatively large damage to the human body
However, the traditional test detection method has small test width, slow speed, low sensitivity, and requires a lot of manpower and material resources

Method used

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  • Screening method for enzyme spectrum of kinase of tumor inhibitor
  • Screening method for enzyme spectrum of kinase of tumor inhibitor

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Embodiment Construction

[0033] The present invention is further illustrated by specific examples below.

[0034] A method for screening tumor inhibitor kinase enzyme spectrum, which comprises the following steps:

[0035] Part 1:

[0036] 1. Determine the highest concentration of the kinase gradient dilution (refer to the relevant report of Invitrogen) and perform the double gradient dilution to obtain several kinase systems with different concentration gradients.

[0037] 2. Mix the kinase into a solution with a final volume of 70uL; add 35ul of buffer to columns 1, 3-12 of the 96-well plate, add 70ul of kinase solution to column 2, and proceed from column 2 to column 11. A total of 10 concentration gradients.

[0038] 3. Transfer the diluted enzyme in the 96-well plate to the 384 plate, 5ul per well, 4 multiple wells (A1, A2, B1, B2, C1, C2, D1, D2, ... B12, C11, C12, D11, D12), centrifuge at 1000 rpm for 20 seconds, and place at room temperature to achieve the highest activity.

[0039] 4. Prepare 420ul of...

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Abstract

The invention relates to C12Q contained enzyme or microorganism measurement or inspection field, and particularly relates to a screening method for an enzyme spectrum of kinase of a tumor inhibitor. The screening method includes the following parts and steps: step one, determining and optimizing the kinase concentration; step two, measuring kinase ATP Km; and step three, measuring IC50 of a positive inhibitor staurosporine or other compounds. With adopting of the above steps, the screening method has the advantages that 66 kinds of kinases can be detected at a time, the detection scale is large, the speed is fast, and the sensitivity is high.

Description

technical field [0001] The invention relates to the field of determination or inspection of C12Q containing enzymes or microorganisms, in particular to a kinase zymogram screening method for tumor suppressors. Background technique [0002] At present, there are two biochemical detection methods for kinase activity: the kinase activity detection method with radioisotopes and the non-radioactive detection method. Among them, the radioisotope-based method has the following disadvantages: a large amount of radioactive waste is produced; the half-life of radioactive ATP is very short, only 14.3 days, so new isotopes need to be ordered frequently; the radioisotope method is not suitable for high-throughput or ultra- High-throughput screening; very strong cross-interference phenomenon; relatively large damage to the human body. [0003] Some new drugs of kinase inhibitors that have been developed have caused huge side effects due to their inhibitory effect on some non-targeted ki...

Claims

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Application Information

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IPC IPC(8): C12Q1/48
Inventor 毛伟峰袁崇刚
Owner EAST CHINA NORMAL UNIV
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